During the 2 years of support of this work by HEW we have by use of model systems demonstrated the feasibility of, and the high precision obtainable with, our new method for the determination of the locations of hydrogen atoms in molecules in crystals. We propose a continuation of the application of this technique to the determination of proton locations in the vicinity of active site prosthetic groups and coordinated metal ions in enzymes in crystals. Such information, which is not available for enzymes from X-ray diffraction, will provide a very useful tool for determining local structure, for measuring changes in conformation of enzyme and substrate which are important for enzymatic action, and for determining the role of bound metals and of hydrogen bonds in enzymatic processes. The development of this method in the case of a molecule such as hen egg white lysozyme for which high resolution X-ray results have accurately fixed the positions of the heavier atoms, will permit its extension to other cases such as human lysozyme and other enzymes for which precise X-ray data are not available. This will then enable one to map the structure of active sites, observe the conformations of bound substrates, and study the enzymatic reaction mechanisms in many physiologically important cases. This new method employs electron paramagnetic resonance and electron nuclear double resonance absorptions of rare earth and other paramagnetic metal ions, bound to enzymes at their active sites in crystals. The anisotropies of the proton-electron hyperfine interactions measured by electron nuclear double resonance yield values of proton coordinates.